Our broad goal is to understand the role of transglutaminase 2 (TG2) in celiac disease pathogenesis. TG2 is known to deamidate dietary gluten peptides, a chemical modification that greatly enhances their recognition by inflammatory, disease-associated T cells. TG2 is also the primary target of autoantibodies in celiac disease. The most significant findings of the past funding cycle were that: (A) extracellula TG2, while ubiquitous in the intestine and most other organs, is held inactive by a C371-372 disulfide bond; and (B) TG2 activity is switched on through contact with thioredoxin (Trx), whose release into the extracellular matrix is promoted by interferon- g. In the course of those studies, we also identified the first small molecule TG2 inhibitor with activity in vivo. Building on these results, our extensively revised application focuses on understanding the biochemical mechanism and physiological relevance of this unusual allosteric regulatory phenomenon. We also propose to exploit this chemical switch to turn TG2 on in a controlled manner, thereby enabling quantitative identification of its endogenous substrates in systems of relevance to celiac disease. Aim 1 proposes to study the mechanistic principles by which Trx recognizes and reduces the C371-C372 disulfide bond in TG2. Three sub-Aims are proposed: (A) Mapping TG2-Trx interactions by alanine-scanning mutagenesis; (B) Engineering a Trx mutant to covalently trap and structurally characterize the transient TG2- Trx complex; and (C) Engineering small molecule inhibitors to inactivate extracellular Trx without affecting its intracellular counterpart. These insights and tools will not only inform our own studies under Aim 2, but will also have broader biological relevance. Aim 2 will test in vivo our hypothesis that Trx activates extracellular TG2 via two complementary approaches. In sub-Aim A, inhibitors from Aim 1C will be tested in one constitutive and two induced animal models where TG2 activity has been observed. In sub-Aim B, covalent TG2-Trx complexes will be trapped and visualized using the Trx mutant from Aim 1B. If successful, our results will substantially strengthen the case for Trx being the missing link between inflammatory T cell activity and TG2 activity in celiac disease. Aim 3 will identify endogenous proteins that undergo post-translational modification when TG2 activity is switched on in the extracellular matrix. In sub-Aim A, we will use cultured enterocytes and fibroblasts to identify such substrates. Sub-Aim 2 seeks to validate their identity in the mouse intestine. Sub-Aim C presents an example of a simple biological assay to understand the role of these post-translational modifications in cell adhesion. These molecular insights are expected to broaden our view of the pathophysiological consequences of up-regulated TG2 activity in celiac disease beyond generation of gluten-derived T cell epitopes.